The present invention is related to a new peroxisome-associated polypeptide, the nucleotide sequence encoding said polypeptide and portions thereof as well as their uses in the diagnosis of several diseases, especially the diagnosis and/or the treatment of lung injuries and diseases, and of oxidative stress-related disorders.
The peroxisomes are organelles nearly ubiquitous in eukaryotic cells. They contain enzymes essential for various catabolic and anabolic pathways. Some of these enzymes are expressed constitutively while others can be induced under appropriate conditions. Peroxisomes carry out a variety of essential reactions such as peroxisomal oxidation and respiration, fatty acid beta-oxidation, cholesterol and dolichol metabolism, ether-phospholipid synthesis, and glyoxylate and pipecolic acid metabolism.
The peroxisomal respiratory pathway is based upon the formation of hydrogen peroxide by a collection of oxidases and the decomposition of the H2O2 by catalase These reactions are responsible for 20% of oxygen consumption in liver, and several oxidases have been identified in peroxisomes. Ethanol elimination via catalase in peroxisomes may be significant in addition to the oxidation via cytosolic alcohol dehydrogenase.
The peroxisomal beta-oxidation system catalyses the beta-oxidative chain shortening of a specific set of compounds which can not be handled by mitochondria: very long chain fatty acids, di- and trihydroxycholestanoic acids, pristanic acid, long chain dicarboxylic acids, several prostaglandins, several leukotrienes, 12- and 15-hydroxyeicosatetraeonic acid, and several mono- and polyunsaturated fatty acids, which are of direct diagnostic relevance for some peroxisomal disorders.
Peroxisomes play also a major role in the synthesis of cholesterol and other isoprenoids. Fibroblasts from patients affected by disorders of peroxisome biogenesis show low capacity to synthesise cholesterol.
Two enzyme activities responsible for introduction of the characteristic ether linkage in ether-linked phospholipids (dihydroacetonephosphate acyltransferase (DHAPAT) and alkyldihydroxyacetonephosphate synthase (alkyl-DHAP synthase)) are localised in peroxisomes. These enzymes are not yet cloned. As demonstrated by the identification of patients with deficiency of either DHAPAT or alkyl-DHAP synthase with severe clinical abnormalities, ether-phospholipids are of major importance in humans.
Peroxisomes are able to detoxify glyoxylate via alanine/glyoxylate aminotransferase. The deficiency of this cloned enzyme causes hyperoxaluria type I. L-pipecolate is a minor metabolite of L-lysine and is catabolised by the L-pipecolate oxidase localised in peroxisomes. The enzyme is deficient in cerebro-hepato-renal (Zellweger) syndrome.
In human, the importance of peroxisomes was emphasised by a number of inherited diseases involving either a defect in the biogenesis of peroxisomes or a deficiency of one (or more) peroxisomal enzymes. So far, 12 different peroxisomal disorders have been described and most of them are lethal.
A wide variety of chemicals have been showed to produce peroxisome proliferation and induction of peroxisomal and microsomal fatty acids-oxidising enzymes activities in rats and mice. Several peroxisomes proliferators have been shown to increase the incidence of liver tumours in these species. Proposed mechanisms of liver tumour formation by peroxisomes proliferators include induction of sustained oxidative stress.
Therefore, newly identified molecules associated with peroxisomes could be used for the development of diagnostic tools and possibly for the improvement of several therapeutical applications of various diseases associated with peroxisomal disorders. In addition, it is useful to identify the molecules present in specific organs like the lung and which may be used as specific markers of inflammatory diseases as well as lung injuries or diseases.
The Inventors have isolated and purified a new sequence of a low molecular weight human broncho-alveolar polypeptide. Said mammal, preferably human, protein or polypeptide (hereafter identified as B18hum protein) has been sequenced and its corresponding genomic DNA (SEQ ID NO 8) and cDNA (SEQ ID NO 1) have been identified. Similarly, the corresponding nucleotide and amino acid sequence from a rat (SEQ ID NO 3 and 4) and from a mouse (SEQ ID NO 5 and 6) have been obtained.
Said sequences present several homologies with other peroxisomal proteins of yeast and comprise a carboxy-terminal tripeptide SQL which is necessary for the specific targeting and translocation of several proteins into the peroxisome.
Therefore, the present invention is related to a new isolated and purified polypeptide sequence having a amino acid sequence which presents more than 70% homology, advantageously more than 85% homology, more preferably more than 95% homology, with the amino acid sequence SEQ ID NO 2. Said amino acid sequence is advantageously obtained from a mammal, preferably from a rat, a mouse or a human.
The present invention is also related to the isolated and purified polypeptide sequence corresponding to the amino acid sequence SEQ ID NO 2 or a portion thereof, preferably an immunoreactive portion (putative immunogenic domain or T or B cell epitopes).
Said portions are advantageously comprised between:
Glutamic acid position 14xe2x80x94Glutamic acid position 28
Alanine position 27xe2x80x94Leucine position 37
Alanine position 43xe2x80x94Glutamic acid position 58
Glutamic acid position 58xe2x80x94Valine position 70
Valine position 81xe2x80x94Leucine position 98
Arginine position 96xe2x80x94Leucine position 113
Serine position 119xe2x80x94Serine position 130
Valine position 138xe2x80x94Threonine position 151
Preferably, said portion has more than 10, 20, 30, 50 or 70 amino acids. Specific portions of the amino acid sequence SEQ ID NO 2 are also portions of more than 70 amino acids which present at least 80% of the proteinic activity (see example 5) of the complete SEQ ID NO 2 sequence. Therefore, the amino acid sequence according to the invention can be partially deleted while maintaining its activity, preferably its anti-oxidative activity, which will be described hereafter.
According to the invention, the amino acid sequence SEQ ID NO 2 presents a pI of 7.16 and a molecular weight of 17047 Dalton as hereafter defined by bidimensional electrophoresis.
The present invention is also related to the nucleotide sequence encoding the amino acid sequence according to the invention and its regulatory sequences upstream said coding sequence. A nucleotide sequence encoding the polypeptide according to the invention is a genomic DNA (see SEQ ID NO 10), a cDNA (see SEQ ID NO 1) or a mRNA, possibly comprising said upstream regulatory sequence. Advantageously, said nucleotide sequence presents more than 70%, advantageously more than 85%, more preferably more than 95% homology with SEQ ID NO 1 or its complementary strand.
According to a preferred embodiment of the present invention, said nucleotide sequence corresponds to the nucleotide sequence SEQ ID NO 1, its complementary strand or a portion thereof.
xe2x80x9cA portion of the nucleotide sequence SEQ ID NO 1xe2x80x9d means any nucleotide sequence of more than 15 base pairs (such as a primer, a probe or an antisense nucleotide sequence) which allow the specific identification, reconstitution or blocking of the complete nucleotide sequence SEQ ID NO 1 (including its regulatory sequences upstream the coding sequence).
Said portions allow the specific identification, reconstitution or blocking by specific hybridisation with the nucleotidic sequence SEQ ID NO 1, preferably under standard stringent conditions, with sequences like probes or primers possibly labelled with a compound (radioactive compound, enzyme, fluorescent marker, etc.), and can be used in a specific diagnostic or dosage method like probe hybridisation (see Sambrook et al., xc2xa7xc2xa79.47-9.51 in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, N.Y. (1989)), genetic amplification (like PCR (U.S. Pat. No. 4,683,195), LCR (Wu et al., Genomics 4, pp. 560-569), CPR (U.S. Pat. No. 5,011,769)).
Exemplary stringent hybridisation conditions are as follows: hybridisation at 42xc2x0 C. in 50% formamide 5xc3x97SSC, 20 mM sodium phosphate, pH 6.8 washing in 0.2xc3x97SSC at 55xc2x0 C. It is understood by those skilled in the art that variation of these conditions occur based on the length and GC nucleotide content of the sequence to be hybridised. Formulas standard in the art are appropriated for determining exact hybridisation conditions (see Sambrook et al.
Preferred examples of said nucleotide portions are as follows:
and the sequences of respectively 601 (SEQ ID NO 8), 604 (SEQ ID NO 9) and 469 (SEQ ID NO 7) base pairs corresponding to specific mRNA alternative splicing of the B18 human nucleotide sequence as described in FIG. 4 (the known genomic sequence incorporating several introns and exons is represented in the sequence SEQ ID NO 10).
Said sequences may be used for a genetic amplification or a probe hybridisation as above-described.
The present invention is also related to a vector comprising the necessary elements for the injection, transfection or transduction of cells and having incorporated one or more of the nucleotide sequences according to the invention. The vector according to the invention is selected from the group consisting of viruses, plasmids, phagemides, cationic vesicles, liposomes or a mixture thereof. Said vector may comprise also one or more adjacent regulatory sequences (such as promoter(s), secretion and termination signal sequence(s)), advantageously operably linked to the nucleotide sequence according to the invention.
The present invention is also related to the cell transformed by said vector and expressing the polypeptide according to the invention.
The nucleotide sequence according to the invention can be also introduced in said cell by the formation of CaPO4-nucleic acid precipitate, DEAE-dextran-nucleic acid complex or by electroporation.
Another aspect of the present invention is related to an inhibitor of the polypeptide according to the invention or the nucleotide sequence according to the invention (including the upstream sequences like promoter-operator regulatory sequence which may be inhibited by a cis- and/or transactivating repressor) . Said inhibitor is advantageously an antibody or a fragment of said antibody such as an hypervariable portion of said antibody directed against the amino acid or nucleotide sequence of the polypeptide according to the invention. Other examples of inhibitors according to the invention are antisense nucleotide sequences which allow the blocking of the expression of the nucleotide sequence according to the invention.
Another aspect of the present invention is related to a diagnostic device (such as a diagnostic kit or a chromatographic column) comprising an element selected from the group consisting of the amino acid sequence of said polypeptide, its nucleotide sequence, and/or the inhibitor according to the invention or a fragment thereof as above-described. Said diagnostic device may comprise also necessary reactants and media for the diagnostic and/or dosage of the nucleotide and/or amino acid sequence of the polypeptide according to the invention, which are based upon the method selected from the group consisting of in situ hybridisation, hybridisation by labelled antibodies, especially RIA (Radio Immuno Assay) or ELISA (Enzymes Linked Immuno-Sorbent Assay) technologies, detection upon filter, upon solid support, in solution, in sandwich, upon gel, dot blot hybridisation, Northern blot hybridisation, Southern blot hybridisation, isotopic or non-isotopic labelling (by immunofluorescence or biotinilised probes), genetic amplification, (especially by PCR or LCR), double immunodiffusion technique, counter-electrophoresis technique, haemagglutination or a mixture thereof.
Another aspect of the present invention concerns a diagnosis method wherein a biological sample from the patient, such as cephalo-rachidian fluid, serum, blood, plasma, urine, broncho-alveolar lavage, stomach lavage, etc., is isolated from the patient, and is put in contact with the diagnostic device according to the invention for the diagnosis or the monitoring of an injury or a disease, preferably a lung injury or an oxidative stress-related disorder, affected by the presence of pro-oxidant agent or oxidative stress such as specific cardio-vascular diseases like arteriosclerosis, neurodegenerative disorders (Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis), apoptosis, inflammatory reactions, allergic reactions such as asthma, hay fever and eczema, high bone mass syndrome, osteopetrosis, osteoporosis-pseudoglioma syndrome, and Bardet-Biedl syndrome 1. Said diagnosis and monitoring upon one or more biological samples obtained from several tissues from the patient can be advantageously obtained by one or more of the methods above-described, which could be adapted according to the specific biological sample by the person skilled in the art.
Therefore, the product according to the invention could be used as a marker for the above-identified injuries, diseases or disorders in a broad spectrum of tissues as shown in the enclosed FIG. 1.
A further aspect of the present invention is related to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an element selected from the group consisting of the nucleotide sequence, the amino acid sequence of the polypeptide according to the invention, the inhibitor directed against said sequences and/or one or more portions thereof.
A last aspect of the present invention is related to the use of the pharmaceutical composition according to the invention for the manufacture of a medicament in the treatment and/or the prevention of lung injuries and/or diseases or of oxidative stress-related disorders.
The present invention is also related to a prevention and/or treatment method of a patient, especially a human patient, preferably affected by lung injuries and/or diseases or by oxidative stress-related disorders, wherein a sufficient amount of the pharmaceutical composition according to the invention is administered to said patient in order to treat, avoid and/or reduce the symptoms of said injuries and/or diseases.
Other injuries and/or diseases which can be prevented and/or treated are injuries and/or diseases affected by the presence of pro-oxidant agents or oxidative stress, such as specific cardio-vascular diseases like arteriosclerosis, neurodegenerative disorders such as Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, apoptosis and inflammatory reactions and some allergic reactions such as asthma, hay fever and eczema, high bone mass syndrome, osteopetrosis, osteoporosis-pseudoglioma syndrome, and Bardet-Biedl syndrome 1.
The pharmaceutically acceptable carrier according to the invention is any compatible non-toxic substance suitable for administering the composition according to the invention to a human patient. Pharmaceutically acceptable carriers according to the invention suitable for oral administration are the ones well known by the person. skilled in the art, such as tablets, coated or non-coated pills, capsules, spray-gas, patches, gels, solutions or syrups. Pharmaceutically acceptable carriers vary according to the mode of administration (intravenous, intramuscular, subcutaneous, parenteral, etc.), and may comprise also adjuvants well known by the person skilled in the art to increase, reduce and/or regulate humoral, local and/or cellular response of the immune system.
The pharmaceutical composition according to the invention may be prepared by the methods, generally applied by the person skilled in the art in the preparation of various pharmaceutical compositions, wherein the percentage of the active compound/pharmaceutically acceptable carrier can vary within very large ranges, only limited by the tolerance of the patient to said pharmaceutical composition, and wherein the limits are particularly determined by the frequency of administration and the possible side-effects of the active compounds or its pharmaceutically acceptable carrier.
Another aspect of the invention is related to the use of the diagnostic device according to the invention for performing upon the patient or upon a biological fluid obtained from the patient, a diagnosis, a dosage and/or a monitoring of the above-mentioned injuries or diseases or oxidative stress-related disorders affecting the patient.
A further aspect of the present invention is related to a cell or a non-human animal, preferably a mammal such as a mouse or a rat, transformed by the vector according to the invention and overexpressing the polypeptide according to the invention, or a non-human animal, preferably a mammal such as a mouse or a rat, genetically modified by a partial or total deletion of its genomic sequence encoding the polypeptide according to the invention (knock-out non-human mammal) and obtained by methods well known by the person skilled in the art such as the one described by Kahn et al. (Cell, Vol. 92, pp. 593-596 (March 1998)).
Other examples of genetically modified non-human animals according to the invention may be a transgenic non-human animal comprising an inhibitor according to the invention, preferably an antisense nucleic acid sequence complementary to the nucleotide sequence according to the invention so placed as to be transcribed into antisense MRNA which is complementary to the nucleotide sequence according to the invention and which hybridises to said nucleotide sequence, thereby reducing or blocking its translation.